Dynamic protein acetylation is essential for normal cell physiology and defects in the enzymes involved are associated with a wide variety of human diseases. The MYST family of histone acetyltransferases are highly conserved, from yeast to man, and they serve as the catalytic subunits of large multi-protein complexes whose structural compositions are also conserved. Aberrant rearrangements or regulation of MYST acetyltransferases are associated with human cancers. However, we know relatively little about their critical protein substrates, the roles they play in the larger protein complexes, or the genetic regulatory pathways that control their function. Recent advances now make tackling these problems feasible and exciting. We are investigating the molecular genetics of two signature members of the MYST family: the Esa1 enzyme of budding yeast, and the Hbo1 enzyme of humans. ESA1 encodes the only essential histone acetyltransferase in budding yeast. It is the catalytic subunit of two multi-protein complexes, NuA4 and picNuA4. We recently made the surprising discovery that catalysis is not the essential function of Esa1, as previously thought. Instead, we propose that Esa1 is a regulatory subunit of NuA4 complexes that uses its catalytic domain as a sensor of physiological signals. We will carry out experiments designed to identify the initiating signals detected by Esa1, and to understand the mechanism of signal transduction through NuA4. We will dissect the molecular genetics of suppressors of esa1 mutations and examine the chromatin changes at promoters of regulated genes in response to esa1 mutations and suppressors. The results of these experiments are poised to completely change the way we think about Esa1 and NuA4 function. Hbo1 is a human MYST family enzyme that serves as the catalytic subunit of multi-protein complexes that include members of the Ing and Jade tumor suppressor families. We discovered that Hbo1 has a causal role in the assembly of the pre-replicative complex for DNA replication licensing. Our results predict that pre-RC proteins may be direct substrates for Hbo1 acetylation. We will carry out experiments to identify sites of lysine acetylation and the molecular mechanism through which they facilitate licensing. We also recently found that tumor suppressor p53 and Hbo1 interact physically and functionally. We propose that this interaction is part of the mechanism that decides between cell division cycle arrest and apoptosis in response to physiological stresses. We will test this hypothesis by carrying out experiments to identify a novel protein substrate of Hbo1 implicated in the pathway and examine its role in regulating pro-apoptotic gene transcription. These experiments will greatly expand our understanding of Hbo1 function in regulating DNA replication and cell proliferation.